Our first efforts are directed against MCD, since this syndrome is characterized by infected B cells that are in the lytic phase of the viral replication cycle. Hence the infected cells are expected to express several KSHV glycoproteins at the surface, including gH/gL, gB, and K8,1A. Though a relatively rare disease particularly with the development of anti-HIV drugs, MCD is associated with a very short life expectancy after diagnosis, and no effective drugs exist. Hence immunotoxins to selectively kill infected B cells may prove efficacious. This work is a continued collaboration with Drs. Corey Casper and Larry Corey who oversee a cohort of MCD patients at the Fred Hutchinson Cancer Center, U. Washington. We previously reported the design and construct of 2 new PE-based immunotoxins derived from newly generated hybridomas against KSHV glycoprotein gH. These were found to potently kill a stable transfectant cell line expressing gH, prompting us to test them against KSHV infected cell lines (BCBL, and Vero-219 harboring latent recombinant KSHV that can be induced by various methods). Since only a small fraction of cells in these lines are in the lytic phase, we developed assays to preferentially test the effects of the immunotoxins. One assay employs quantitative PCR to measure the number of virions released by the lytic-phase cells. Unfortunately, we found that the anti-gH immuntoxins were minimally efficacious in reducing the output of infectious progeny. This may be due to the fact that duriing activation from latency, gH expression is reduced relatively late;however K8.1A, a surface glycoprotein not directly involved in fusion/entry, is expressed much earlier on infected cells. Hence we began a collaboration with Dr. Bala Chandran to generate new immunotoxins from anti-K8.1A hybridomas previously described by his group. We have cloned the V regions of the corresponding heavy and light chains, and are in the process of linkiing these to the effector domains of PE to generate new KSHV-targeted immmunotxoins. Another approach involves generating immunotoxins agaist the KSHV-encoded viral G protein-coupled receptor (vGPCR), which has been shown to be critical for the induction and maintainence of KS tumors, despite its expression in only a fraction cells within the tumor. We are continuing efforts to generate MAbs against the vGPCR for immunotoxin production. As an alternative "proof-of-concept" approach, we have produced targeted toxins containing specific chemokines (IL-8) as targeting moieties. While these cannot be used clinically because they will also kill normal cells expressing the corresponding chemokine receptors (mainly neutrophils for IL-8), they can be tested in a model system developed by Silvio Gutkind (NIDCR) in which cells expressing vGPCR are coinjected into immunodeficiency mice along with an exceess of cells expressing other KSHV latent genes;the tumors contain the same low proportion of vGPCR-expressing cells as the injected input, but the tumor can be eliminated by treatment with an agent that the vGPCR-cells are selectivey sensitized to. Hence we can test whether the chemokine-toxin effectively eliminates these tumors. Positive results would encourage further development mAbs against the vGPCR for immunotoxin production and testing.